1. Field of the Invention
A simple and convenient method for concentrating a biomolecule, including protein or nucleic acid molecules, from a sample. Purified and isolated biomolecules obtained by this method. Methods for improving the specificity or sensitivity of detecting a biomolecule by concentration and/or purification or isolation of the biomolecule according to the method of the invention.
2. Description of the Related Art
The investigation of diagnostically relevant biological samples such as serum, plasma, blood, swab samples or organ triturations for detection of infectious pathogens has gained enormously in importance in recent years. Virus infections such as HIV, HCV or HBV are on the increase worldwide.
Commercially available methods for detecting viral nucleic acids are based on known nucleic acid amplification techniques such as PCR (polymerase chain reaction), real time PCR, NASBA (nucleic acid sequence-based amplification) or branched DNA detection. However, the known detection methods are pushing the limits of detection sensitivity, especially in the testing of large-volume samples (such as pools of 96 individual plasma samples). The problem lies in the fact that the commercial tests used always need isolation of the nucleic acids (for example, of viral type). Isolation of viral nucleic acids is usually performed from liquid samples with a volume of 50-200 μL.
In order to be able to achieve higher detection sensitivity or to be able to investigate even pooled individual samples more sensitively, methods for working up large-volume samples are needed. Since this is not possible with the common nucleic acid isolation methods, attempts have been made to concentrate virus particles of a sample in a first step. One possibility for concentrating viral particles consists in ultracentrifuging the sample to be investigated. In this way virus particles accumulate on the bottom of the test tube. This method depends on an ultracentrifuge, and is also time-consuming and unsuitable for routine diagnostics. Alternative methods consist in precipitation of virus particles by means of polyethylene glycol/sodium chloride, followed by centrifugation (Yamamoto et al., Virology 40 (1970) 734; Morandi et al., J. Clin. Microbiol. 36 (1998) 1543-1538). In these cases, various mixtures of PEG and sodium chloride are used and these reagents are mixed with the biological sample. Thereafter the mixture is incubated for a lengthy time in the cold and then the virus (protein)-NaCl/PEG precipitates are obtained by centrifugation. These methods are also laborious and need much time. Another problem is the further processing of the precipitates for isolation of the viral nucleic acids. In many cases the precipitates can be redissolved only with great difficulty. This greatly influences the efficiency and quality of nucleic acid isolation. DE Patent 19856415 C2 describes a method that includes the known NaCl/PEG precipitation, after which the nucleic acids are isolated in a manner known in itself by binding on a silicate solid phase. The extent to which this method is superior to the adequately known method of NaCl/PEG precipitation with the known problems is not clear. Furthermore, this method also needs incubation in the cold and twenty minutes of centrifugation.
The method supposedly makes it possible to isolate viral nucleic acids from a sample of up to 10 mL. A further commercially available variant, which supposedly permits processing up to 1-mL samples, is based on concentration of the viral nucleic acids using a special detergent. An initial incubation of the sample with a lysis reagent then leads to lysis of the viruses. Thereafter a “detergent-nucleic acid complex” is formed. The mixture is centrifuged and the pellet obtained is then treated by proteolysis, after which the nucleic acid is again isolated in a manner known in itself via binding on a silicate solid phase (QIAamp UltraSens Virus Handbook). In this case also; problems with resuspension of the pellet are mentioned. Furthermore, the method permits processing only of samples with a volume of at most 1 mL.
The content of unexamined disclosure WO 03/095079 A2 is a filter membrane based on completely polymerized alginate fibers as well as further support materials for concentrating and/or separating components from liquids. The components that are supposedly concentrated are protozoans. The technique is oriented toward the field of determination and concentration of microbiological components in drinking water, etc. What is described therein is a method for producing a special membrane layer (fleece) on the basis of a polyuronic acid polymer. A filter membrane is produced in a laborious process and then used for specific purposes. In the cited method, a liquid is passaged over a previously prepared filter membrane based on a polyuronic acid polymer. The cited disclosure does not give any information on how to dissolve the polymer in order to achieve subsequent isolation of nucleic acids from the biological sample or how to isolate a nucleic acid from viruses after they have been concentrated. Furthermore, there is no information on how viruses can be concentrated with the membrane, since the concentration principle is based on the fact that the protozoans are concentrated in well-defined pores formed in the polyuronic acid polymer. Isolation of nucleic acids from biological samples is neither described nor made obvious in WO 03/095079 A2. Furthermore, there is indication that the polymer is dissolved with a chaotropic buffer.
U.S. Pat. No. 5,739,019 A describes the isolation of microorganisms from an aqueous sample. The patent does not describe a method in which biomolecules are concentrated from a biological sample in polymers based on polyuronic acid, after which nucleic acids are isolated, but instead discloses a mechanism whereby polyuronic acid can be polymerized by addition of calcium chloride. The cited patent utilizes this mechanism, in which aqueous solution of potassium chloride and microorganisms is mixed dropwise with a polyuronic acid, and the microorganisms are then encapsulated in these droplets. These encapsulated microorganisms are then cultivated in so-called selective growth media in subsequent steps of the method. What is not done is subsequent isolation of nucleic acids from the biomolecules for diagnostic detection. No mention is made of the isolation of DNA.
The isolation of a virus is reported in Tribune de Cebedeau, 1976, 29 (390), pp. 186-94. Isolation of a nucleic acid is not mentioned. A membrane is produced in known manner by means of alginate and calcium chloride. A solution from which the viruses are supposedly adsorbed on the filter or in the filter is then passaged via this membrane filter (polymerized alginate filter system). Thus the method is used to concentrate viruses on or in membranes (or more correctly hydrogels) by filtering the liquid containing the viruses through this alginate membrane.
The described prior art methods clearly show that the processing of large-volume samples is still inherently complicated and associated with considerable disadvantages, as is also an increase of the volume of a sample in order to increase the sensitivity for detection of viral nucleic acids.
Heretofore it has not been described in the prior art that nucleic acids can be bound on or in polymerized alginate and then recovered. Heretofore it has also not been known that microorganisms (especially viruses) can be concentrated in order to isolate nucleic acids therefrom and in turn to detect the viruses after concentration.